The present invention relates generally to Polymerase Chain Reaction systems, and more particularly to systems and methods for removing step discontinuities in Polymerase Chain Reaction data.
The Polymerase Chain Reaction (PCR) is an in vitro method for enzymatically synthesizing or amplifying defined nucleic acid sequences. The reaction typically uses two oligonucleotide primers that hybridize to opposite strands and flank a template or target DNA sequence that is to be amplified. Elongation of the primers is catalyzed by a heat-stable DNA polymerase. A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by the polymerase results in an exponential accumulation of a specific DNA fragment. Fluorescent probes or markers are typically used in the process to facilitate detection and quantification of the amplification process.
A typical real-time PCR curve is shown in FIG. 1, where fluorescence intensity values are plotted vs. cycle number for a typical PCR process. In this case, the formation of PCR products is monitored in each cycle of the PCR process. The amplification is usually measured in thermocyclers which include components and devices for measuring fluorescence signals during the amplification reaction. An example of such a thermocycler is the Roche Diagnostics LightCycler (Cat. No. 20110468). The amplification products are, for example, detected by means of fluorescent labelled hybridization probes which only emit fluorescence signals when they are bound to the target nucleic acid or in certain cases also by means of fluorescent dyes that bind to double-stranded DNA.
The fluorescence dyes used in real-time PCR are temperature sensitive. The thermal-cycling profile may include changing the annealing temperature. The change in annealing temperature manifests as a discontinuity in the fluorescence read-out magnitude. FIG. 2 shows an illustrative case where a change in annealing temperature can be seen after cycle 6.
The fluorescence data read-out corresponding to an experiment conducted using the profile of FIG. 2 is shown in FIG. 3. It can be clearly seen in the HEX channel that a discontinuity is present between cycle 5 and 6. FIG. 3 is taken from a B19 Parvo-virus sample with 104 IU/ml concentration. The FAM dye being less sensitive to temperature has a jump discontinuity of a lower magnitude than the HEX dye.
However, if the concentration of the target increases, it is possible that the discontinuity may not be reflected in fluorescence data. For example, considering a B19 Parvo-virus sample with a concentration of 2.9×1011 IU/ml, the discontinuity is not seen in FIG. 4a even though there was a change in annealing temperature after cycle 5.
Some current PCR systems implement methods for removal of a step discontinuity due to changes in the annealing temperature. Such systems typically require assay specific input parameters for the cycle number corresponding to the change in annealing temperature and the maximum fluorescence change expected. Such methods have a number of limitations. First, they are unable to handle high titer samples. This can be seen in FIG. 4b, where the B19 Parvo sample at 2.9×1011 IU/ml concentration has been incorrectly handled by such a system. Second, such methods may only handle a small maximum (e.g., five) absolute fluorescence units change at the point of discontinuity. It has been observed in multiple assays that the magnitude of fluorescence change at the discontinuity varies widely. Setting up an assay specific input to indicate maximum change is difficult to optimize. Third, such methods may be unable to handle fluorescence data where the baseline drift is high. In such cases, the correction provided may be inadequate. Fourth, if a spike happens to be present at a distance of one cycle or less from the cycle number corresponding to the change in annealing temperature, then the correction provided has been known to cause changes in reported result concentrations; i.e., a wrong result.
Therefore it is desirable to provide systems and methods for processing sigmoid-type or growth curves, and PCR curves in particular, which overcome the above and other problems. In particular, the systems and methods should implement temperature step correction in a manner that is reliable and robust.